The purpose of this research is to understand cytoskeletal organization and functions during cell division of the intracellular protozoan parasite Toxoplasma gondii, a model for harmful apicomplexan parasites that include Plasmodium falciparum and Cryptosporidium parvum. Despite the importance of the cytoskeleton for cell division in these unicellular organisms the mechanisms that power the separation of the genome remain uncertain, largely because only limited data exist about the organization and composition of the cytoskeleton. Recent data suggest an unconventional division machinery that divides the nucleus and separates the apicoplast, a single non-photosynthetic plastid that is essential for parasite survival. We hypothesize (1) that the parasite's unconventional division apparatus is based on a complex organization of microtubules, their organizing center (the centriole-centrosome complex) and perhaps other cytoskeletal components yet to be determined. Fibers of the division apparatus may associate with the pellicle (the parasite's outer membrane system) to separate the genome and the apicoplast during cell division in T. gondii; we hypothesize (2) that the centriole-centrosome complex plays an important role in the organization of the cytoskeleton and may contain proteins that differ from mammalian cells. Our specific aims are to (1) analyze the parasite's cytoskeleton during cell division with focus on the interactions with the pellicle, the apicoplast and the nucleus, and (2) to characterize the centriole-centrosome complex with focus on proteins that may differ from mammalian cells and may serve as targets to inhibit cell division in apicomplexan parasites. The novel approach of the proposed experiments is to use high-resolution field emission scanning electron microscopy (HRFESEM) on thick-sectioned resin de-embedded material. This method used in preliminary experiments allowed for the first time viewing of actin-like filaments and intermediate-like filaments which had not been possible with any other method available so far. We will further isolate the division apparatus of T. gondii and generate antibodies to determine parasite-specific centrosome proteins. This research will address unresolved questions regarding the cytoskeleton and the centriole-centrosome complex during cell division in T. gondii and, perhaps, the existence of other cytoskeletal fibers. This research will advance our understanding of cell division in apicomplexan parasites and may lead to the identification of targets to control replication of harmful apicomplexan parasites. Pilot data from this RO3 research will be used to apply for funding through the RO1 mechanism.